Higher Animal Cells Research Articles

HCN channels are essential for the escape response of Paramecium.

When mechanical stimulation was applied to free swimming Paramecium, forward swimming velocity transiently increased due to activation of the posterior mechanosensory channels. The behavior response, known as "escape response," requires membrane hyperpolarization and the activation of K-channel type adenylate cyclases. Our hypothesis is that this escape response also involves activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. HCN channels are activated by hyperpolarization and are modulated by cyclic nucleotides such as cAMP and cGMP. They play a critical role in many excitable cells in higher animals. If HCN channels act in Paramecium, this should help to enhance and prolong hyperpolarization, thereby increasing the swimming speed of Paramecium. This study used RNAi to examine the role of the HCN channel 1 in the escape responses by generating hcn1-gene knockdown cells (hcn1-KD). These cells showed reduced mechanically-stimulated escape responses and a lack of cGMP-dependent increases in swimming speed. Electrophysiological experiments demonstrated reduced hyperpolarization upon injection of large negative currents in hcn1-KD cells. This is consistent with a decrease in HCN1 channel activity and changes in the escape response. These findings suggest that HCN1 channels are K+ channels that regulate the escape response of Paramecium by amplifying the hyperpolarizations elicited by posterior mechanical stimulation.

A Theoretical Study on the Cell Differentiation Forming Stem Cells in Higher Animals

The recent genome sequencing of multicellular diploid eukaryotes reveals an enlarged repertoire of protein genes for signal transmission but it is still difficult to elucidate the network of signal transmission to drive the life cycle of such an eukaryote only from biochemical and genetic studies. In the present paper, a theoretical study is carried out for the cell differentiation, the formation of stem cells and the growth from a child to the adult in the higher animal. With the intercellular and intracellular signal transmission in mind, the cell differentiation is theoretically derived from the process by the transition of proliferated cells from proliferation mode to differentiation mode and by both the long-range interaction between distinctive types of cells and the short-range interaction between the same types of cells. As the hierarchy of cell differentiation is advanced, the original types of self-reproducible cells are replaced by the self-reproducible cells returned from the cells differentiated already. The latter type of self-reproducible cells are marked with the signal specific to the preceding differentiation and become the stem cells for the next stage of cell differentiation. This situation is realized under the condition that the differentiation of cells occurs immediately after their proliferation in the development. The presence of stem cells in the respective lineages of differentiated cells strongly suggests another signal transmission for the growth of a child to a definite size of adult that the proliferation of stem cells in one lineage is activated by the signal from the differentiated cells in the other lineage(s) and is suppressed by the signal from the differentiated cells in its own lineage. This style of signal transmission also explains the metamorphosis and maturation of germ cells in higher animals.

Histochemical localization of N-acetylhexosamine-binding lectin HOL-18 in Halichondria okadai (Japanese black sponge), and its antimicrobial and cytotoxic anticancer effects

We studied localization and physiological activities of a lectin showing specific binding to N-acetylhexosamines, termed HOL-18, purified from Japanese black sponge (Halichondria okadai). Antiserum against the lectin was generated in rabbit and applied for immunohistochemical analyses. HOL-18 was expressed specifically around water pores and on spicules of sponge tissues. It showed strong binding to a variety of N-acetylhexosamines: N-acetyl D-glucosamine, N-acetyl D-galactosamine, N-acetyl mannosamine, N-acetyl muramic acid, and N-acetyl neuraminic acid. Hemagglutination induced by the lectin was inhibited by lipopolysaccharides and a peptidoglycan. HOL-18 inhibited growth of a gram-positive bacterium (Listeria monocytogenes), gram-negative bacteria (Escherichia coli, Shigella boydii, Pseudomonas aeruginosa), and a fungus (Aspergillus niger). It displayed anti-biofilm activity against P. aeruginosa. HOL-18 was internalized into conidiophores of A. niger, and displayed notable antifungal activity. Fluorescence microscopy revealed binding and incorporation of the lectin into human cancer cell lines HeLa, MCF-7, and T47D, but not Caco-2. HOL-18 displayed dose-dependent cytotoxic effects against HeLa, MCF-7, and T47D, with respective IC50 values 40, 52, and 63 μg/mL. In HeLa cells, it activated phosphorylation of MAPK pathway molecule (ERK1/2) and activated caspase-3 to trigger apoptosis. HOL-18 thus has the potential to upregulate metabolic pathways in higher animal cells through binding to N-acetylhexosamines.

001 Milestones in cryobiology

Cryobiology is a discipline that has its origins in experiments that were published exactly 350 years ago. In 1663, Henry Power, a British physician, reported that he had used a mixture of snow and salt to freeze nematodes. When he thawed the samples three hours later, he stated that “all my little Animals made their re-appearance and danced and frisked about as lively as ever.” Over the centuries, this discipline has evolved and grown into a formal science that ranges from theoretical analysis of the behavior of solutions and the response of biological cells at subzero temperatures to application of very practical procedures to cryopreserve 250 million doses of cattle sperm annually as well as sperm of more than 60 species of mammals, fish and birds. Cryobiology now plays a critically important role in human and animal medicine, agriculture, research and as a fundamental discipline itself. Its applications are so diverse that it is not possible to even mention all of the milestones that have been achieved. Throughout the 18th century, scientists studied the phenomenon of cryptobiosis, the interruption of normal metabolism of cells and simple organisms by cooling them to low temperatures. By the 19th century, such studies had developed into formal experimental biology in which systematic measurements of the effects of freezing were made. By the 1920s and 30s, investigators were attempting to preserve cells of many animal species by cooling them to temperatures as low as that of liquid nitrogen and even liquid helium. In some isolated cases, cells of various types survived; for example, Jahnel in Germany observed motile human spermatozoa that had been cooled to −269 °C. In the 1940s and 1950s, several observations significant enough to be called milestones had been made. These included the demonstration by Polge and his colleagues that glycerol protected rooster spermatozoa against the damaging effects of freezing. Within a short time, this finding had been extended to cattle and human spermatozoa and then to erythrocytes. Freezing of sperm resulted in live births of calves and children. To search for possible causes of injury, Luyet and Meryman independently studied formation and re-crystallization of ice crystals in aqueous solutions. Lovelock began to apply formal physical–chemical analysis to the subject. Other biologists measured effects of freezing and thawing on a wide variety of microorganisms and animal cells. In the 1950s, there was sufficient interest in these observations of freezing to justify convening several symposia. In the 1960s, Mazur formulated his mathematical analysis of the response of cells when they are frozen. Shortly thereafter, it was demonstrated that cells of higher animals subjected to freezing and thawing exhibited responses very similar to those of microorganisms. This, in turn, led to the successful cryopreservation of numerous types of animal and plant cells. In the 1970s, application of fundamental principles resulted in the successful cryopreservation of mouse embryos and then to those of more than 25 other species. Then, procedures were devised to cryopreserve organized tissues of many types and from diverse species. Now, in the 21st century, an incredible variety of cells and tissues of plants and animals are routinely preserved not only by freezing but also by vitrification and even by drying. Many of these achievements were first attempted by the earliest investigators of cryptobiosis. Source of funding: None declared. Conflict of interest: None declared. sleibo@uno.edu

Growth and Lactogenic Hormones and Adaptive Immunocompetence

Adaptive immune reactions are based on lymphocyte proliferation, and are regulated by mechanisms which are involved in growth regulation of all cells in higher animals. Growth and lactogenic hormones (GLH) or type I cytokines (TICTK) are of fundamental importance for the development and function of the immune system and indeed for the entire organism. GLH/TICTK deliver the competence signal first to lymphocytes and other cells, which enable the cells to receive stromal or adherence signals that decide what the cell will do next, proliferate, differentiate, or function. This group of signals control cell growth, is delivered by cell-to-cell and cell-to-matrix signalling. Adhesion molecules, tissue bound hormones, cytokines and matrix components mediate these signals. The antigen receptor belongs to the Immunoglobulin Family of adhesion molecules. Hence the antigen signal is capable to activate specifically the immune response, it can inhibit it also. Within the immune system antigen presentation represents the adhesion signal for which cell-to-cell interaction by adhesion molecules is obligatory. Adhesion molecules are fundamental to the organization of multi-cellular organisms and the signals delivered by them serve the basis of species, organ and tissue specific recognition. This recognition system has been perfected during evolution from self-recognition to individually specific antigen recognition. This system also plays a role in the elimination of degenerated and neoplastic cells. Cell-to-cell signaling has a dominant power over other signals to commit the cell to proliferation. The mitotic cycle is then completed by cytokine signals. Cytokines are tissue hormones which are usually, but not always, secreted by the same cells that deliver the second signal. IGF-I and insulin fulfil frequently this role, but cytokines may also deliver this third signal. The nature and combination of these three groups of signals will determine the fate of each cell, which may be survival, proliferation, differentiation and function or alternately apoptosis. Hormones and neurotransmitters, that alter signal delivery, modulate further this basic pattern of animal cell growth. It is concluded that GLH/TICTK maintain immunocompetence, which enables the immune system to respond to specific antigenic and mitogenic stimuli. Competence signalling comes from either the CNS or by TICTK, which is an autologous signal by the immune system. This dual signalling secures that the immune system is capable of surviving major disasters in the personal history of the host. Indeed memory T and B ells survive autonomously, and live trough major catastrophic events, and when the disaster is over, memory cells regenerate the adaptive immune system and bring back homeostasis of immune function which is the prerequisite for healing and homeostasis of the entire organism. The hypothalamic hormone, vasopressin (VP) regulates healing and recovery from disease.

The cell cycle, including the mitotic cycle and organelle division cycles, as revealed by cytological observations

It is generally believed that the cell cycle consists essentially of the mitotic cycle, which involves mitosis and cytokinesis. These processes are becoming increasingly well understood at the molecular level. However, successful cell reproduction requires duplication and segregation (inheritance) of all of the cellular contents, including not only the cell-nuclear genome but also intracellular organelles. Eukaryotic cells contain at least three types of double membrane-bounded organelles (cell nucleus, mitochondria and plastids), four types of single membrane-bounded organelles (endoplasmic reticulum, Golgi apparatus, lysosomes and microbodies) and the cytoskeleton, which comprises tubulin-based structures (including microtubules, centrosome and spindle) and actin microfilaments. These membrane-bounded organelles cannot be formed de novo and daughter organelles must be inherited from parent organelles during cell cycle. Regulation of organelle division and its coordination with the progression of the cell cycle involves a sequence of events that are subjected to precise spatio-temporal control. Considering that the cells of higher animals and plants contain many organelles which tend to behave somewhat randomly, there is little information concerning the division and inheritance of these double- and single-membrane-bounded organelles during the cell cycle. Here, we summarize the current cytological and morphological knowledge of the cell cycle, including the division cycles of seven membrane-bounded and some non-membrane-bounded organelles. The underlying mechanisms and the biological relevance of these processes are discussed, particularly with respect to cells of the primitive alga Cyanidioschyzon merolae that have a minimum of organelles. We discuss unsolved problems and future perspectives opened by recent studies.

Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting

In higher animal cells, the principal limitation of gene-targeting technology is the extremely low efficiency of targeted integration, which occurs three to four orders of magnitude less frequently than random integration. Assuming that random integration mechanistically involves non-homologous end-joining (NHEJ), inactivation of this pathway should reduce random integration and may enhance gene targeting. To test this possibility, we examined the frequencies of random and targeted integration in NHEJ-deficient chicken DT40 and human Nalm-6 cell lines. As expected, loss of NHEJ resulted in drastically reduced random integration in DT40 cells. Unexpectedly, however, this was not the case for Nalm-6 cells, indicating that NHEJ is not the sole mechanism of random integration. Nevertheless, we present evidence that NHEJ inactivation can lead to enhanced gene targeting through a reduction of random integration and/or an increase in targeted integration by homologous recombination. Most intriguingly, our results show that, in the absence of functional NHEJ, random integration of targeting vectors occurs more frequently than non-targeting vectors (harboring no or little homology to the host genome), implying that suppression of NHEJ-independent random integration events is needed to greatly enhance gene targeting in animal cells.

Study on the mutations in the D-loop region of mitochondrial DNA in cervical carcinoma

Study on genesis and development of tumor is mainly concentrated on gene mutation in nucleus. But in recent years, the role of mitochondrial DNA (mtDNA) mutation in tumor genesis has been given more attention, which is the only extra-nucleus DNA in cells of higher animals. Carcinoma of the uterine cervix is a common tumor in gynecology. There are few reports of mtDNA mutation in this area. The focus of this study was to investigate the mitochondrial DNA mutation in tumor tissues of cervical carcinomas patients and their relationship to tumorigenesis and tumor development. The D-loop region of 24 cervical carcinomas together with the adjacent normal tissues were amplified by PCR and sequenced. Among the 24 cervical carcinomas, 30 mutations were identified with the mutations rate of 37.5% (9/24).There were eight microsatellite instabilities among the mutations and 13 new polymorphisms which were not reported previously in the GeneBank. The D-loop region of mitochondrial DNA is a highly polymorphoric and mutable region and the mutations rate is relatively high in patients with cervical carcinomas.

Getting a grip on calcium regulation

Temporally and spatially regulated Ca2+ signals control numerous physiological processes. In most cells in higher animals, the plasma membrane (PM) Na/Ca exchanger (NCX) helps manage the cytosolic Ca2+ concentration ([Ca2+]CYT) and Ca2+ stored in the sarco-/endoplasmic reticulum (S/ER), thereby influencing Ca2+ signaling (1–3). This tightly regulated transporter can move one Ca2+ ion either out of or into cells in exchange for three Na+. The direction of net Ca2+ movement depends on the prevailing membrane potential (V M) as well as the Na+ and Ca2+ concentration gradients because there is net charge transfer. Cardiac myocytes have large dynamic swings in V M and [Ca2+]CYT during each heartbeat, and NCX plays an especially interesting role: It can mediate Ca2+ entry during the upstroke of the action potential, help maintain the elevated [Ca2+]CYT and contraction during the action potential plateau, and then extrude Ca2+ during repolarization and diastole (1). It is noteworthy that NCX is regulated by cytosolic Na+ and Ca2+ at sites that do not directly participate in the ion translocation (4, 5). A rise in cytosolic Na+ rapidly stimulates and then inactivates the exchanger (6); in contrast, cytosolic Ca2+ activates the exchanger and relieves the Na+-dependent inactivation (4, 5). Two elegant studies by Philipson, Abramson, and colleagues, including one in this issue of PNAS (7), provide novel insight into how Ca2+ binds to and alters the conformation of the Ca2+ regulatory sites in the cardiac/neuronal NCX (NCX type-1) (7, 8).

The mechanisms of nitric oxide production from exogenous and endogenous NO-donating compounds in cells of higher animals and the influence of nitric oxide on deoxyribonucleotide and DNA synthesis

The mechanisms of exogenous nitric oxide (NO) production through in vivo biotransformation of nitro-, nitroso-and amino-containing substances were discussed. In addition, the mechanisms of production and cellular sources of endogenous NO, appearing in the blood and tissues after the exposure to various DNA-damaging factors, have been considered. Considerable quantities of endogenous NO were detected in the body in the first hours after translation inhibition by cycloheximide or animal exposure to superlethal radiation doses, i.e., after the exposure to factors inducing destructive processes. The time and dose dependences of exogenous and endogenous NO production have been established. NO produced after a single or repeated administration of NO-donating compounds as well as endogenous NO proved to inhibit deoxyribonucleotide (dNTP) and DNA synthesis in animal tissues. Nonspecific compensatory responses to disturbed protein homeostasis included cyclic production of endogenous NO. The maximum levels of nitrosyl complexes were registered when the rate of protein synthesis decreased. The role of polyamines in the induction of macromolecule biosynthesis is discussed and NO production from these arginine-rich compounds is proposed. NO is released at the stage of polyamine inactivation. The inactivation mechanism includes the hydroxylation of aminogroups by NO synthase, the formation of nitroso intermediates, and their denitrosation with NO release.

Is aging the price for memory?

Aging (senescence) is apparent in animals that possess long-lived postmitotic cells but is negligible in primitive species, such as hydras and other Cnidarians, all of whose cells are constantly renewed by cell division. This repetitive mitotic activity precludes the progressive intracellular accumulation of damaged biomolecules and organelles, which are obvious concomitants of aging in neurons and other long-lived cells of higher animals. We assume that the development of long-lived postmitotic cells, now found in the overwhelming majority of species, represented a useful evolutionary change. Probably, of particular importance was the evolution of long-lived neurons, which are required for long-term memory. However, the appearance of long-lived postmitotic cells not only increased fitness, but also gave rise to the aging process.

The role of nucleophosmin in centrosome duplication.

In higher animal cells, duplication of centrosomes is triggered by CDK2/cyclin E-mediated phosphorylation. Nucleophosmin (NPM)/B23, a multifunctional protein, has recently been identified as one of the substrates of CDK2/cyclin E in centrosome duplication. Centrosome-bound NPM/B23 dissociates from centrosome upon phosphorylation by CDK2/cyclin E, which in turn triggers initiation of centriole duplication. Duplicated centrosomes remain free of NPM/B23 till mitosis. When the nuclear membrane breaks down during mitosis, NPM/B23 re-localizes to centrosomes. Upon cytokinesis, each daughter cell receives one centrosome bound by NPM/B23, which again dissociates from the centrosome upon exposure to CDK2/cyclin E at mid-late G1 phase of the next cell cycle. Thus, NPM/B23 would constitute one of the licensing systems for centrosome duplication, ensuring the coordination of centrosome and DNA duplication, which limiting duplication once per cell cycle.

A Mammalian In Vitro Centriole Duplication System: Evidence for Involvement of CDK2/Cyclin E and Nucleophosmin/B23 in Centrosome Duplication

Centrosome duplication in mammalian cells is a highly regulated process, occurs in coordination of other cell cycle events. However, molecular exploration of this important cellular process had been difficult due to unavailability of a simple assay system. Here, using centrosomes loosely associated with nuclei isolated from cultured cells, we developed a cell-free centriole (duplication unit of the centrosome) duplication system: unduplicated centrosomes bound to the nuclei are able to undergo duplication in the presence of G1/S extracts. We show that the ability of G1/S extracts to induce centriole duplication in vitro depends on the presence of active CDK2/cyclin E. It has been shown that dissociation of centrosomal nucleophosmin (NPM)/B23 triggered by CDK2/cyclin E-mediated phosphorylation is required for initiation of centrosome duplication. We show that centriole duplication is blocked when nuclei were preincubated with the anti-NPM/B23 antibody that prevents phosphorylation of NPM/B23 by CDK2/cyclin E. These studies provide not only direct evidence for the requirement of CDK2/cyclin E and phosphorylation of NPM/B23 for centrosomes to initiate duplication, but a valuable experimental system for further exploration of the molecular regulation of centrosome duplication in somatic cells of higher animals.

Molecular Cloning and Characterization of Two Isoforms of Saccharomyces cerevisiae Acyl-CoA:Sterol Acyltransferase

Esterification of cholesterol by acyl-CoA:cholesterol acyltransferase (ACAT) is a key element in maintaining cholesterol homeostasis in cells of higher animals. In the budding yeast, Saccharomyces cerevisiae, accumulation of ergosteryl esters accompanies entry into stationary phase and sporulation. We have determined that two genes in yeast, SAT1 and SAT2, encode isozymes of acyl-CoA:sterol acyltransferase (ASAT) which are functionally related to ACAT. The SAT1 isozyme is the major catalytic isoform, accounting for at least 65-75% of total ASAT activity. Targeted deletions of one or both genes do not compromise mitotic cell growth or spore germination. However, diploids that are homozygous for a SAT1 null mutation exhibit significantly reduced sporulation efficiency. Furthermore, a larger fraction of the sporulating diploids arrest after the first meiotic division. Human ACAT expressed in sat1 sat2 mutant cells can catalyze esterification of cholesterol and, to a lesser extent, ergosterol in vitro, but restores ergosteryl oleate formation in vivo to only approximately 8% of that catalyzed by yeast ASAT in wild-type cells.

Dynamics of Tetrahymena macronuclear lamina during cell division

During mitosis, the nuclear lamina in higher eukaryotic cells undergoes a distinctly morphological change. It breaks down into lamin polymers or monomers at prophase. At telophase, the lamins reassemble around the condensed chromatin to form the layer of lamina. Using antiserum to mammalian lamins, we studied the dynamics of lamina during cell division in the macronuleus of Tetrahymena shanghaiensis, which divided in the way of amitosis. In contrast to those in higher animal cells, the typical perinuclear lamin distribution in the macronucleus persisted throughout the whole cell cycle. It was further found that in some synchronized cells, the lamin distribution displayed an unusual pattern consisting of a series of spots within the macronucleus. Using South-western hybridization, we found that the purified 66 KD lamin in Tetrahymena showed specific affinity with the telomere DNA sequence in the same species. Therefore, we propose that pattern of immunofluorescence may be due to the interaction of lamin protein with the nucleoli and the condensed chromatins in the macronucleus.

Movement of axoplasmic organelles on actin filaments from skeletal muscle

It was recently shown that, in addition to the well-established microtubule-dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments [Kuznetsov et al., 1992, Nature 356:722-725]. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video-enhanced contrast differential interference contrast (AVEC-DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine-phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC-DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments.

Actin-dependent organelle movement in squid axoplasm.

Studies of organelle movement in axoplasm extruded from the squid giant axon have led to the basic discoveries of microtubule-dependent organelle motility and the characterization of the microtubule-based motor proteins kinesin and cytoplasmic dynein. Rapid organelle movement in higher animal cells, especially in neurons, is considered to be microtubule-based. The role of actin filaments, which are also abundant in axonal cytoplasm, has remained unclear. The inhibition of organelle movement in axoplasm by actin-binding proteins such as DNase I, gelsolin and synapsin I has been attributed to their ability to disorganize the microtubule domains where most of the actin-filaments are located. Here we provide evidence of a new type of organelle movement in squid axoplasm which is independent of both microtubules and microtubule-based motors. This movement is ATP-dependent, unidirectional, actin-dependent, and probably generated by a myosin-like motor. These results demonstrate that an actomyosin-like mechanism can be directly involved in the generation of rapid organelle transport in nerve cells.

The Immune Response to Nematode Parasites: Modulation of Mast Cell Numbers and Function During Strongyloides Stercoralis Infections in Nonhuman Primates

Mucosal mast cell numbers are modulated in the intestines of rodents during parasitic infections. These mast cells can degranulate in response to worm antigens, and this event has been suggested to play a protective role for the host. To examine whether mast cells in higher animals play a role in protecting from disseminated parasitic disease, mast cell numbers and responsiveness to parasite antigens were evaluated in 5 Erythrocebus patas infected with the human intestinal nematode Strongyloides stercoralis. Initial infection and subsequent challenge infections were associated with increase in jejunal histamine and mast cell numbers, and these mast cells could release histamine in response to parasite antigens. Jejunal mast cell numbers returned to normal during a chronic phase of infection. The cells lost their ability to respond to antigenic stimulation following limited steroid treatment. Subsequent activation of chronic infections to fatal disseminated disease by more prolonged steroid treatment was associated with a marked decrease in jejunal mast cell numbers and histamine. In one animal which succumbed to severe disease without steroid treatment, jejunal mast cells were refractory to worm antigens.

Ionic currents of smooth muscle cells isolated from the ctenophoreMnemiopsis

The ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis were examined by using conventional two-electrode voltage clamp and whole-cell patch clamping methods. Several separable currents were identified. These include: (1) a transient and (2) a steady-state voltage-activated inward current; both are tetrodotoxin (TTX) and saxitoxin (STX) insensitive, partly reduced by decreasing external Ca2+ or Na+ or by addition of 5 mM Co2+, D-600 or verapamil and are totally blocked with 5 mM Cd2+; (3) an early, transient, cation-dependent, outward K+ current (IKCa/Na); (4) a transient, voltage-activated, outward K+ current provisionally identified as IA; (5) a delayed, steady-state, voltage-activated outward K+ current (IK) and (6) a late, transient, outward K+ current which is blocked by Cd2+ and evident only during long voltage pulses. Despite their phylogenic origin, most of these currents are similar to currents identified in many vertebrate smooth and cardiac muscle preparations, and other excitable cells in higher animals.

Control of phytopathogens by inhibitors of polyamine biosynthesis.

It is now clear that polyamines (PAs) are required for the normal growth and development of microorganisms as well as cells of higher animals and plants,1, 2, 3. In all organisms, putrescine (Put) is formed from ornithine via ornithine decarboxylase (ODC); in some microorganisms and in higher plants, Put may also be formed from arginine via arginine decarboxylase (ADC). Put is then transformed to the triamine spermidine (Spd) and the tetramine spermine (Spm) by successive enzyme-mediated aminopropyl transfers from S-adenosylmethionine (SAM).KeywordsPowdery MildewStem RustVerticillium WiltOrnithine DecarboxylasePolyamine BiosynthesisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.